RESUMO
OBJECTIVES AND BACKGROUND: The involvement of DNA methylation in periodontal disease is not clear. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis is involved in the progression of periodontal disease. We recently developed an in vitro model of LPS infection in human periodontal fibroblast cells (HPdLFs) for a prolonged period. In this study, we examined genome-wide analysis of DNA methylation in HPdLFs stimulated with LPS derived from P. gingivalis for a prolonged period. We noted the hypermethylation of extracellular matrix (ECM)-related genes and examined whether hypermethylation affected their transcription levels. MATERIAL AND METHODS: HPdLFs were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The culture was repeated, alternating 3 d with LPS derived from P. gingivalis and 3 d without LPS for 1 mo. Untreated samples were used as controls. DNA was analyzed using the human CpG island microarray. Quantitative methylation-specific polymerase chain reaction was carried out to confirm reproducibility of the microarray data. The expression levels of mRNA of the selected ECM-related genes from the data were analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: We found 25 ECM-related genes with hypermethylation at the CpG island of the promoter region, which exhibited a fourfold greater hypermethylation than controls. Among these genes, hypermethylation of nine ECM-related genes, FANK1, COL4A1-A2, 12A1 and 15A1, LAMA5 and B1, MMP25, POMT1 and EMILIN3, induced a significantly downregulated expression of their mRNA. CONCLUSION: These results indicate that LPS derived from P. gingivalis may cause DNA hypermethylation of some ECM-related genes followed by downregulated expression of their transcriptional levels.
Assuntos
Metilação de DNA , Matriz Extracelular/genética , Fibroblastos/metabolismo , Lipopolissacarídeos/farmacologia , Porphyromonas gingivalis , Células Cultivadas , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Transcrição GênicaRESUMO
To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in the cytoplasm of the treated fibroblasts. In contrast, the other cytochalasins, including cytochalasin B, cytochalasin C, cytochalasin D, and cytochalasin H, were found to induce the formation of nuclear rodlets. Both cytoplasmic and nuclear rodlets found in the cytochalasin-treated cells were similar in ultrastructures to those induced by 5 to 10 percent (vol/vol) dimethyl sulfoxide in the same type of cells.
Assuntos
Actinas/metabolismo , Citocalasinas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Capeamento Imunológico/efeitos dos fármacos , Camundongos , ViscosidadeRESUMO
We successfully detected dengue virus (DENV) genome in urine and saliva but not in plasma samples from a Japanese dengue fever patient. The results of the present study suggest that detection of DENV genome in urine and saliva can be an effective diagnostic method, particularly for children with viral hemorrhage.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Adulto , Dengue/urina , Vírus da Dengue/genética , Feminino , Genoma Viral , Humanos , Saliva/virologiaRESUMO
The nucleotide sequence of nuclear 5.4 S RNA, a new species of small nuclear RNA (snRNA) of mouse cells, was determined. The 5.4 S RNA consists of 138 nucleotide residues containing 1 mol each of 2,2,7- trimethylguanosine (m3(2,2,7) G), 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um) and pseudouridine as modified nucleosides. This RNA has a cap structure, m3(2,2,7) ++GpppAm -, at its 5'-terminus and sequences complementary to the terminal consensus sequences of introns. The sequence complementary to the 5'-splice junction, A-U-C-C-psi-U-A-C-C-U-G, is very similar to the 5'-terminal sequence of U1 RNA.
Assuntos
RNA/genética , Animais , Sequência de Bases , Linhagem Celular , Leucemia Experimental , Camundongos , Conformação de Ácido Nucleico , Purinas/análise , Pirimidinas/análise , RNA/isolamento & purificação , Capuzes de RNA/isolamento & purificação , Splicing de RNA , RNA Nuclear Pequeno , Ribonuclease T1 , Ribonuclease PancreáticoRESUMO
Cytosine residues of nucleic acids were converted to 4-thiouracil residues with hydrogen sulfide in pyridine and water to examine the secondary and tertiary structures of mouse 5 S rRNA. The cytosine residues at positions 10, 24, 34 (or 36), 39, 44 (or 46) and 63 were converted preferentially when the treatment was carried out at 28 degrees C. This result supports the model of the secondary structure of 5 S rRNA of Nishikawa, K. and Takemura, S. ((1974) FEBS Lett. 40, 106-109) consisting of five helices and five loops. As the temperature was increased to 35 degrees C, additional cytosine residues in positions 26, 52 and 78 were modified to moderate extents.
Assuntos
Citosina , Sulfeto de Hidrogênio , RNA Ribossômico , Tiouracila , Animais , Sequência de Bases , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Rim , Camundongos , Camundongos Endogâmicos C3H , RibonucleasesRESUMO
Chemical modification of mouse 5 S rRNA with kethoxal was carried out to examine the secondary structure. The guanine residues located at positions 37, 41, 56, 66, 75 and 89 were modified. The relative rates of reaction are in the order G37, G56, G89, G66, G41, G75 at 28 degrees C and G37, G41, G56, G89, G75, G66 at 35 degrees C. These results support a secondary structure model containing 5 helices and 5 loops and indicate that the region around position 37 is the most exposed in higher-order structure.
Assuntos
Aldeídos/farmacologia , RNA Ribossômico/metabolismo , Animais , Sequência de Bases , Butanonas , Guanina/metabolismo , Metilação , Camundongos , Peso Molecular , Conformação de Ácido Nucleico , Ribonuclease T1RESUMO
Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.
Assuntos
Incisivo/embriologia , NF-kappa B/fisiologia , Odontogênese/fisiologia , Germe de Dente/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Ameloblastos/citologia , Amelogenina/análise , Animais , Apoptose/fisiologia , Proteínas Morfogenéticas Ósseas/genética , Esmalte Dentário/citologia , Epitélio/embriologia , Proteínas Hedgehog/fisiologia , Quinase I-kappa B/fisiologia , Imageamento Tridimensional/métodos , Incisivo/anormalidades , Queratina-15/genética , Camundongos , Camundongos Mutantes , Microrradiografia/métodos , Mutação/genética , Receptores Patched , Fenótipo , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/fisiologia , Germe de Dente/anormalidades , Dente Supranumerário/etiologia , Dente Supranumerário/genética , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Microtomografia por Raio-X/métodosRESUMO
We previously reported that a host cell glycoprotein, gp180, binds duck hepatitis B virus particles, and is encoded by a member of the carboxypeptidase gene family (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022-15028). After that report, carboxypeptidase D (CPD) was subsequently purified from bovine pituitary and characterized as a novel carboxypeptidase E (CPE)-like enzyme, with many characteristics in common with duck gp180 (Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007-25013). CPD is now supposed to play an important role in a secretory pathway. To clarify the function of gp180 further, we have isolated and analyzed human and mouse homologues of duck gp180. cDNA clones derived from human HepG2 cells and mouse livers have been isolated on the basis of homology to the duck gp180. The suggested open reading frames of the human and mouse cDNA encode 1380 and 1377 amino acid proteins, respectively and have three carboxypeptidase homologous domains (A, B, and C). Domains A and B have completely conserved the residues known to have the enzymatic activity of carboxypeptidase, but domain C in each cDNA does not. Northern blotting revealed a ubiquitous tissue distribution of human gp180 mRNA with several transcript species. Expression of human gp180 cDNA in transfected 293T
Assuntos
Carboxipeptidases/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Fígado/enzimologia , Glicoproteínas de Membrana/genética , Proteínas , Sequência de Aminoácidos , Animais , Sequência de Bases , Carboxipeptidases/biossíntese , Carboxipeptidases/química , Carcinoma Hepatocelular , Bovinos , Centrômero/genética , Clonagem Molecular , Patos , Biblioteca Gênica , Vírus da Hepatite B do Pato/fisiologia , Humanos , Neoplasias Hepáticas , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
On RPC-5 column chromatography, the main valine acceptor activity of tRNA (tRNA2Val) from rat ascites hepatoma cells was eluted later than that of normal rat liver tRNA (tRNA1Val). The tRNA2Val was aminoacylated by E. coli amino-acyl-tRNA synthetase, while tRNA1Val from normal rat liver was not. Rat fetal liver tRNAVal was also aminoacylated by E. coli aminoacyl-tRNA synthetase. tRNA1Val (rat liver) and tRNA2Val (ascites hepatoma) were each purified to a homogeneous state by RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis, and their sequences were determined by post-labeling techniques. Ascites hepatoma tRNA2Val differed from rat liver tRNA1Val in that Gm18, C32 and an unknown modified nucleoside, N34, in the latter tRNA were mostly replaced by G, Cm, and inosine, respectively. In addition, 3'-terminal adenosine was not present in tRNA1Val (normal rat liver), but was in tRNA2Val (ascites hepatoma). Other modifications and the primary structures of the two tRNAValS were found to be the same. Thus it was concluded that the new iso-acceptor species of tRNA Val in ascites hepatoma cells is due to a change of post-transcriptional modification, not to a change of tRNA transcription. The unique feature of the change of post-transcriptional modification in tRNA2Val (ascites hepatoma) is that both hypo- and hyper-modification take place simultaneously in the tRNA molecule depending the locations of nucleotide residues.